The long term objectives of this project are to understand how changes in protein glycosylation regulate antibody dependent immune responses, and then to utilize that knowledge to develop improved antibody-based therapies for diseases such as cancer. The specific aims of this project are to: 1. Develop methods to produce homogeneous glycoproteins for the study and optimization of antibody glycosylation. 2. Determine the role of N-linked glycosylation in IgG Fc interactions with Fc receptors. 3. Develop methods to incorporate site-specific chemical modifications onto glycosylated IgG Fcs to facilitate biochemical studies and to modify bioactivity. 4. Determine the role of N-linked glycosylation in regulating antibody dependent cellular immune responses. As part of this research, novel methods will be developed to produce homogeneously glycosylated antibody fragments of the immunoglobulin G (IgG) subclasses, and also to attach small molecule receptor ligands to those antibody fragments site-selectively. The glycosylated and chemically modified antibody fragments thus produced will be utilized in in vitro biochemical and structural studies of antibody interactions with Fc receptors to determine the role of glycosylation and IgG subclass in regulating cellular immune responses. Antibody fragments attached to receptor ligands will be utilized to target cancer cells for antibody dependent immune responses in antibody dependent cell-mediated cytotoxicity (ADCC) assays and complement dependent cytotoxicity (CDC) assays. These cell-based assays will be used to optimize antibody dependent immune responses directed against target cells. In the future the knowledge gained by these experiments and the techniques developed will be valuable in developing antibody-based protein therapeutics directed against cancer and other diseases. PUBLIC HEALTH RELEVANCE: Antibodies are important therapeutic proteins that are used to treat cancers, arthritis, and many other diseases. The goal of this project is to understand how modification of human antibodies with sugars regulates antibody dependent immune responses, and then use the knowledge gained from these studies to develop better antibody-based treatments for diseases.